Development of a High‐throughput Fluorescent‐Based Assay to Assess Cytochrome P450 CYP3A7 Activity in Neonatal Human Liver Microsomes

Abstract

Cytochromes P450 (CYPs) are a superfamily of human enzymes essential for the metabolism of drugs, xenobiotics, and many endogenous hormones such as steroids. In adults, CYP3A4 is the primary xenobiotic metabolizing enzyme expressed in the liver and intestine, and its metabolism of xenobiotics has been extensively studied over the past several decades. In contrast, CYP3A7, the 3A sub-family enzyme predominantly expressed in neonates and developing infants, has been less studied and has no standard or efficient high-throughput screening (HTS) method to determine its enzymatic activity. As more pharmaceutical agents are being developed to specifically target this fragile patient population, a robust and reliable HTS assay is needed in order to facilitate pre-clinical testing of drug candidates for both safety and efficacy. Additionally, development of such an assay would be extremely beneficial to identify potential drug-drug interactions that might occur before actual administration of the drugs. In this study, our goal was to identify an efficient, cost-effective fluorescent substrate specific for CYP3A7 and develop an HTS assay supporting it. To that end, the fluorescent substrates resorufin benzyl ether, nile red, 7-benzyloxy-4-trifluoromethylcoumarin, dibenzylfluorescein, 7-benzyloxymethyloxy-3-cyanocoumarin, 7-benzyloxyquinoline, and dibenzyloxymethylfluorescein were examined and their metabolite formation by CYP3A7 was quantified using fitted fluorescent spectra. The maximum rates of reactions (Vmax) were determined for each substrate, along with the corresponding Michaelis-Menten constant (Km). Other characteristics, such as the Z-score and signal-to-noise ratio, were assessed to further demonstrate the utility of this methodology to screen for CYP3A7 activity in a high-throughput fashion. These findings will help determine which substrate is most useful for studies in heterogenous systems (i.e. human liver microsomes) and drug screening.